Structural features of a three-stranded DNA junction containing a C-C junctional bulge.

We have examined the stability of junctional base pairs in a three-way DNA junction with two unpaired cytidine residues at the branch point using two-dimensional nuclear Overhauser effect spectroscopy in H2O solution. Our data directly support the presence of two of the three junctional Watson-Crick base pairs, with indirect support for the third as well. These results complement the data presented in the preceding paper, where we examined the nonexchangeable proton resonance assignments of three-way DNA junctions from NOESY data in D2O solution. We have incorporated the NOE data from both sets of experiments, using this information as input for a combined distance geometry (DG) and simulated annealing (SA) protocol designed to derive three-dimensional structures of the junction molecule consistent with the NMR data. Although the data does not allow us to derive a unique solution for the structure of the molecule, certain conformational features are invariably present in our models. We demonstrate the existence of a preferred, pair-wise stacking arrangement between two of the three helices in the junction. Furthermore, the remaining duplex stem is situated so that it always forms an acute angle with just one of the arms from the quasi-continuous helix. The unpaired residues provide an extended backbone segment linking two of the helices together. The first unpaired base on the 5' end loops out from the interior of the molecule to reside along the minor groove of one helix. The second is located within the interior of the molecule, stacking below one of the junctional base pairs. Our findings suggest that junctional base pair stacking is an important determinant in the conformation of multistranded nucleic acid junctions. In three-way junctions, the presence of unpaired bases at the branch point provides a relief from covalent constraints that would otherwise prevent the simultaneous realization of both base pairing and base pair stacking within the branch point of the molecule.